KR102507311B1 - Use of siRNA specific for protein S in the treatment of hemophilia - Google Patents

Abstract

The present invention provides siRNA against protein S for use in methods of treating hemophilia. A method of treating hemophilia in a patient in need thereof, comprising administering to the patient a molecule comprising the siRNA according to the present invention, and a dosage form for preventing or treating hemophilia comprising the molecule comprising the siRNA according to the present invention. within the scope of the present invention.

Description

Use of siRNA specific for protein S in the treatment of hemophilia

The present invention relates to the treatment of hemophilia using siRNA directed against protein S.

background of invention

Hemophilia A (HA) and B (HB) are genetic X-linked disorders. These are caused by mutations in the factor VIII (FVIII) (F8) or factor IX (FIX) genes (F9), respectively, resulting in a deficiency of the encoded protein that is an essential component of the intrinsic coagulation pathway (FIG. 1A).

Severe hemophilia patients often suffer from spontaneous bleeding within the musculoskeletal system, such as myositis. If left untreated, it can lead to disability at a young age.

Current hemophilia treatment includes factor replacement therapy. Although this therapy improves quality of life (QoL), it still has some drawbacks. The factor is administered intravenously, and because of its short half-life, it must be injected repeatedly, causing great discomfort to the patient, and there is a risk of infection and venous damage. More importantly, patients under factor replacement therapy may develop inhibitory alloantibodies. Inhibitors render replacement therapies ineffective and limit patients' access to safe and effective standards of care, increasing patients' risk of morbidity and mortality.

New therapies focus on the development of products that can potentially improve adherence to therapy and QoL by reducing the frequency of prophylactic infusions. In addition to the long-acting FVIII and FIX, novel approaches are strategies to rebalance coagulation in patients with hemophilia, including replacement of genes required for the production of endogenous clotting factors, bispecific antibody technology that mimics the coagulation function of defective factors, and tissue factors including targeting of coagulation inhibitors such as pathway inhibitors (TFPI) or antithrombin. Recently, it has been shown that activated protein C (APC)-specific serpins rescue thrombin generation in vitro and restore hemostasis in a hemophilia mouse model.

Based on the above state of the art, an object of the present invention is to provide means and methods for providing a new hemophilia treatment. This object is achieved by the claims of this specification.

description of the invention

A first aspect of the present invention provides siRNA against protein S for use in a method of treating hemophilia.

In the context of this specification, the term hemophilia relates to a condition in which the body's ability to form clots is impaired. Genetic forms of hemophilia include the genetic disorders hemophilia A, hemophilia B and hemophilia C.

Protein S in the context of this specification relates to human "vitamin K-dependent protein S" (UniProt ID P07225) encoded by gene PROS1 (NCBI Gene ID: 5627).

There are two transcript variants of human protein S. Transcript variant 2 has no alternating in-frame exons in the 5' coding region compared to transcript variant 1. Encoded isoform 2 is shorter than isoform 1. Transcript variant 2 is characterized by SEQ ID NO: 001. Transcript variant 1 is characterized by SEQ ID NO: 002.

In the context of this specification, the term siRNA (small/short interfering RNA) refers to an RNA molecule capable of interfering with (in other words: inhibiting) the expression of a gene containing a nucleic acid sequence complementary to the sequence of the siRNA in a process called RNA interference. It is about. The term siRNA is meant to include both single-stranded siRNA and double-stranded siRNA. SiRNAs are generally characterized by a length of 17-24 bp. Double-stranded siRNAs are derived from longer double-stranded RNA molecules (dsRNA). Long dsRNAs are cleaved by endo-ribonuclease (called Dicer) to form double-stranded siRNAs. In the nucleoprotein complex (referred to as RISC), double-stranded siRNAs are unwound to form single-stranded siRNAs. RNA interference often acts through the binding of a siRNA molecule to an mRNA molecule with a complementary sequence, resulting in mRNA degradation. RNA interference is also possible by binding of siRNA molecules to intronic sequences of pre-mRNA (immature, unspliced mRNA) in the nucleus of cells, degrading the pre-mRNA.

We investigated whether the target protein S (PS) could promote hemostasis in hemophilia by reregulating coagulation (Fig. 1B). The PS encoded by the PROS1 gene acts as a cofactor for APC in the inactivation of factors Va (FVa) and FVIIIa and for TFPI in the inhibition of FXa. This dual role makes PS a major regulator of thrombin generation.

In certain embodiments, siRNA comprises 17-24 nucleotides.

In certain embodiments, siRNA comprises 18-22 nucleotides.

In certain embodiments, siRNA comprises 19 nucleotides.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 002.

In certain embodiments, the siRNA is SEQ ID NO: 003, SEQ ID NO: 004, SEQ ID NO: 005, SEQ ID NO: 006, SEQ ID NO: 007, SEQ ID NO: 008, SEQ ID NO: 009, SEQ ID NO: 010, SEQ ID NO: 011, SEQ ID NO: 012, SEQ ID NO: 012 013, SEQ ID NO: 014, SEQ ID NO: 015, SEQ ID NO: 016, SEQ ID NO: 017, and SEQ ID NO: 018.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 003.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 004.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 005.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 006.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 007.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 008.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 009.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 010.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 011.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 012.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 013.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 014.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 015.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 016.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 017.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to SEQ ID NO: 018.

In certain embodiments, siRNAs are characterized by sequences that are reverse complementary to sequences formed by juxtaposing any two contiguous sequences of the sequences defined above. As a non-limiting example, the siRNA is characterized by a sequence that is reverse complementary to a sequence formed by juxtaposing SEQ ID NO: 014 and SEQ ID NO: 015.

In certain embodiments, the siRNA is a group consisting of SEQ ID NO: 014 (exon 12), SEQ ID NO: 011 (exon 9), SEQ ID NO: 006 (exon 4), SEQ ID NO: 012 (exon 10), and SEQ ID NO: 013 (exon 11). It is characterized by a sequence that is reverse complementary to a sequence selected from .

In certain embodiments, the siRNA is nucleotides 1-100, 101-200, 201-300, 301-400, 301-400, 401-500, 501-600, 601-700, 701-800, 801 of SEQ ID NO: 001. -900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1501-1600, 1701-1800, 1801-1900, 1901-2000, 2001-2100, 2101-20201, 2001-20201 , 2301-2400, 2501-2600, 2701-2800, 2801-2900, 2901-3000, 3001-3100, 3101-3200, 3201-3300, 3301-3400, 3401-3500 or 3501-3580. characterized by complementary sequences.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1-100 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 101-200 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 201-300 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 301-400 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 401-500 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 501-600 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 601-700 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 701-800 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 801-900 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 901-1000 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1001-1100 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1100-1200 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1201-1300 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1301-1400 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1401-1500 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1501-1600 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1601-1700 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1701-1800 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1801-1900 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 1901-2000 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2001-2100 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2100-2200 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2201-2300 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2301-2400 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2401-2500 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2501-2600 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2601-2700 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2701-2800 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2801-2900 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 2901-3000 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3001-3100 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3100-3200 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3201-3300 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3301-3400 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3401-3500 of SEQ ID NO: 001.

In certain embodiments, the siRNA is characterized by a sequence reverse complementary to the sequence characterized by nucleotides 3501-3580 of SEQ ID NO: 001.

In certain embodiments, siRNAs are characterized by sequences that are reverse complementary to sequences formed by juxtaposing any two contiguous sequences of the sequences defined above. As a non-limiting example, the siRNA is characterized by a sequence reverse complementary to a sequence formed by juxtaposing 1501-1600 of SEQ ID NO: 001 and 1501-1600 of SEQ ID NO: 001.

In certain embodiments, the siRNA is directed against an intronic sequence of protein S.

In certain embodiments, the siRNA is a group consisting of SEQ ID NO: 014 (exon 12), SEQ ID NO: 011 (exon 9), SEQ ID NO: 006 (exon 4), SEQ ID NO: 012 (exon 10), and SEQ ID NO: 013 (exon 11). It is characterized by a sequence that is reverse complementary to a sequence selected from .

In certain embodiments, the siRNA is SEQ ID NO: 019 (siRNA_1), SEQ ID NO: 020 (siRNA_2), SEQ ID NO: 021 (siRNA_3), SEQ ID NO: 022 (siRNA_4), SEQ ID NO: 023 (siRNA_5), SEQ ID NO: 024 (siRNA_6), SEQ ID NO: 025 (siRNA_7), SEQ ID NO: 026 (siRNA_8), SEQ ID NO: 027 (siRNA_9), SEQ ID NO: 028 (siRNA_10), SEQ ID NO: 029 (siRNA_11), SEQ ID NO: 030 (siRNA_12), SEQ ID NO: 031 (siRNA_13), sequence SEQ ID NO: 032 (siRNA_14), SEQ ID NO: 033 (siRNA_15), SEQ ID NO: 034 (siRNA_16), SEQ ID NO: 035 (siRNA_17), SEQ ID NO: 036 (siRNA_18), SEQ ID NO: 037 (siRNA_19), SEQ ID NO: 038 (siRNA_20), SEQ ID NO 039 (siRNA_21), SEQ ID NO: 040 (siRNA_22), SEQ ID NO: 041 (siRNA_23), SEQ ID NO: 042 (siRNA_24), SEQ ID NO: 043 (siRNA_25), SEQ ID NO: 044 (siRNA_26), SEQ ID NO: 045 (siRNA_27), SEQ ID NO: 046 (siRNA_28), SEQ ID NO: 047 (siRNA_29), SEQ ID NO: 048 (siRNA_30), SEQ ID NO: 049 (siRNA_31), SEQ ID NO: 050 (siRNA_32), SEQ ID NO: 051 (siRNA_33), SEQ ID NO: 052 (siRNA_34), SEQ ID NO: 053 ( siRNA_35), SEQ ID NO: 054 (siRNA_36), SEQ ID NO: 055 (siRNA_37), SEQ ID NO: 056 (siRNA_38), SEQ ID NO: 057 (siRNA_39), SEQ ID NO: 058 (siRNA_40), SEQ ID NO: 059 (siRNA_41), SEQ ID NO: 060 (siRNA_42 ), SEQ ID NO: 061 (siRNA_43), SEQ ID NO: 062 (siRNA_44), SEQ ID NO: 063 (siRNA_45), sequence A sequence comprising or consisting of a sequence selected from the group comprising SEQ ID NO: 064 (siRNA_46), SEQ ID NO: 065 (siRNA_47), SEQ ID NO: 066 (siRNA_48), SEQ ID NO: 067 (siRNA_49), and SEQ ID NO: 068 (siRNA_50) characterized by

In certain embodiments, siRNAs are provided for use in a method of treating hemophilia A.

In certain embodiments, siRNAs are provided for use in a method of treating hemophilia B.

Similarly, within the scope of the present invention is a method of treating hemophilia in a patient in need thereof comprising administering to the patient a molecule comprising a siRNA according to the present invention.

Similarly, a dosage form for preventing or treating hemophilia comprising a molecule comprising a siRNA according to the present invention is provided.

In the context of this specification, the expression “a molecule comprising the siRNA according to the present invention” refers to siRNA and molecules according to the present invention, from which the siRNA according to the present invention can be produced in mammalian cells by the RNA interference pathway. , in particular dsRNA molecules.

A "nucleotide" in the context of the present invention is a nucleic acid or nucleic acid analog building block, the oligomers of which are capable of forming selective hybrids with RNA oligomers on the basis of base pairing. In this context, the term nucleotide is derived from the classical ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, and the classical deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine. Deine and deoxycytidine. It is also analogous to nucleic acids, such as phosphothioate, 2'O-methylphosphothioate, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkages, nucleobases of glycine attached to the alpha-carbon) or locked nucleic acids (LNA; 2'O, 4'C methylene bridged RNA building blocks). The hybridization sequence may consist of any of the above nucleotides, or mixtures thereof.

In certain embodiments, a hybridization sequence of a siRNA according to the invention comprises 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides.

In certain embodiments, the hybridization sequence is at least 95% identical, more preferably at least 96%, 97%, 98%, 99% or 100% identical to the reverse complementary sequence of SEQ ID NO: 001 or SEQ ID NO: 002. In certain embodiments, the hybridization sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, LNA and/or PNA nucleotides or mixtures thereof.

In the context of this specification, the terms sequence identity and percent sequence identity refer to values determined by comparing two aligned sequences. Methods for aligning sequences for comparison are well known in the art. Alignment of sequences for comparison can be found in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) local homology algorithm, Needleman and Wunsch, J. Mol. Biol. 48:443 (1970) Global Sorting Algorithm, Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or computer implementation of these algorithms, including but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyzes is publicly available, eg, through the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/).

One example of comparing amino acid sequences is the BLASTP algorithm, which uses the following default settings: Expected Threshold: 10; word size: 3; max match in query range: 　0; Matrix: BLOSUM62; Gap Coast: Existence 11, Expansion 1; Configuration Adjustment: Conditional configuration score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm using default settings: expected threshold: 10; word size: 28; max match in query range: 　0; Match/Disagree Score: 　1.-2; Gap cost: linear. Sequence identity values provided herein, unless otherwise stated, are derived from the BLAST suite program (Altschul et al., J. Mol. Biol. 215:403-410) using the basic parameters identified above for protein and nucleic acid comparisons, respectively. (1990)).

In some embodiments, the hybridization sequence comprises ribonucleotides, phosphothioates and/or 2'-O-methyl-modified phosphothioate ribonucleotides.

In some embodiments, the hybridization sequence comprises deoxynucleotides, phosphothioate deoxynucleotides, phosphothioate ribonucleotides and/or 2'-O-methyl-modified phosphothioate ribonucleotides.

Where alternatives to single separable features are presented herein as “embodiments,” it is to be understood that such alternatives may be freely combined to form individual embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which additional embodiments and advantages may be obtained. These examples are intended to illustrate the present invention and do not limit the scope of the present invention.

Description of the drawing
1 shows that loss of X-ase activity rescues Pros1−/− mice. A, Schematic diagram of thrombin generation in hemophilic condition. One of the major coagulation complexes is the intrinsic tenase (X-ase) complex. X-ases include activated FIX (FIXa) as a protease, activated FVIII (FVIIIa) as a cofactor and factor X (FX) as a substrate. Although the production or exposure of tissue factor (TF) at the site of injury is the key event initiating coagulation via the extrinsic pathway, the endogenous pathway X-ase is important due to the limited amount of active TF available in vivo and activates FX (FXa). ), the presence of TFPI inhibits the TF/activator VII (FVIIa) complex (Fig. 1a). Thus, sustained thrombin generation is dependent on the activation of FIX and FVIII (Figure 1a). FVIII is activated by both FXa and thrombin, and FIX is activated by both FVIIa and activator XI (FXIa), a process amplified since the latter was previously activated by thrombin. Consequently, progressive increases in FVIII and FIX activation occur when FXa and thrombin are formed, and targeting Pros1 is an experimental approach to enhance thrombin generation in severe hemophilia A and B. Assessment of DIC blood parameters in hemophilic adult mice with or without CD, Pros1 deficiency: F8 ^-/- Pros1 ^+/+ , F8 ^-/- Pros1 ^+/- and F8 ^-/- Pros1 ^{-/ -} (C) , and F9 ^-/- Pros1 ^+/+ , F9 ^-/- Pros1 ^+/- and F9 ^-/- Pros1 ^{-/ - PS} (antigenicity), FVIII (coagulability) in adult mice (D) ( n =5/group) activity) or FIX (coagulation activity) plasma levels; Platelet ( n =7/group), fibrinogen ( n =8/group), PT ( n =6/group) and TAT ( n =6/group) in hemophilia A group (c); and platelets ( n =5/group), fibrinogen ( n =4/group), PT ( n =4/group) and TAT ( n =4/group) in hemophilia B group (D). EF, macroscopic images of lungs from F8-/-Pros1-/- mice 24 hours after a single intravenous injection of 2U/g recombinant FVIII (Advate® infusion (E) and corresponding microscopic evaluation for fibrin clots in lung areas ( F).G, F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^{- /-} recombinant FVIII (Advate® Administration: 5 iv injections of 0.3 U/g Advate® (Time of injection: 1 hour before catheterization and 1 hour, 4 hours, 8 hours and 1 hour after catheterization) 16 hours) (n=3) (G, white and black columns) and 24 hours ( n =3) after a single iv injection at F8 ^-/- Pros1 ^{- /-} (G, dotted column) fibrinogen and plasma levels of TAT, and after repeated iv injections of 0.3 U/g Advate® (H) and after single iv injections of 0.3 U/g Advate®iv (i) in F8 ^-/- Pros1 ^{- /-} at 24 hours Representative immunohistochemistry capable of detecting fibrin clots in lung and liver areas.All data are expressed as mean ± sem; ns, not significant; *, P <0.05 **; P <0.005.
Figure 2 shows a murine thrombosis model. TF-induced venous thromboembolism in AC, F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ , F8 ^{-/ -} Pros1 ^+/- and F8 ^-/- Pros1 ^-/- mice ( n = 10/ genotype). Anesthetized mice were intravenously injected via the inferior vena cava with different doses of recombinant TF (Innovin): ½ dilution in A (˜4.3 nM TF) and ¼ dilution in BC (˜2.1 nM TF). In (A), a group of F8 ^+/+ Pros1 ^+/+ mice received an injection of low molecular weight heparin (Enoxaparin 60 μg/g sc). The time to onset of respiratory arrest lasting at least 2 min was recorded. The experiment was terminated at 20 minutes. Kaplan-Meier survival curves (AB). C, 2 min after the onset of respiratory arrest or upon completion of the 20 min observation period, lungs were excised and examined for fibrin clots (immunostaining for insoluble fibrin, mAb clone 102-10). D, F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ and thrombi formation in FeCl ₃ -injured mesenteric arteries recorded by biomicroscopy in F8 ^-/- Pros1 ^-/- mice, representative experiments ( n =3/genotype). D, F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ and thrombi formation in FeCl ₃ -injured mesenteric arteries recorded by biomicroscopy in F8 ^-/- Pros1 ^-/- mice, representative experiments ( n =3/genotype).
Figure 3 shows a tail bleeding model. Blood was collected after 2 mm (A) and 4 mm (B) tail incisions for 30 min (A) and 10 min (B) in fresh saline tubes; Total blood loss (μl) was then determined. F8 ^+/- Pros1 ^+/+ and F8 ^+/+ Pros1 ^+/+ mice (white columns) served as controls (n=5 for all groups in A, n=6 for all groups in B). C, Anti-human PS antibody altered tail hemorrhage after 4 mm incision.
4 shows an acute cirrhosis model. A, Knee diameter difference between 72 h post-injury and pre-injury in F8 ^-/- Pros1 ^+/+ , F8 ^-/ - Pros1 ^+/- , F8 ^-/- Pros1 ^-/- and F8 ^+/+ Pros1 ^+/+ mice . B, Microscopic evaluation of the intra-articular space of the knee of representative uninjured and injured limbs after 72 h in F8 ^+/+ Pros1 +/ ⁺ , F8 - ^/ - Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- mice ( Mason's trichrome staining and immunostaining for insoluble fibrin). C, mPS silencing in vivo using specific siRNA: evaluation of joint diameter 72 h post injury in F8 ^-/- Pros1 ^+/- and F8 ^-/- Pros1 ^+/+ mice treated with a single ip injection of mPS siRNA or control siRNA. D, Microscopic evaluation of the intra-articular space of the knee of a representative injured leg after 72 h in F8 ^−/− Pros1 ^+/+ mice pre-treated with mPS siRNA or control siRNA (Masson's trichrome staining). Measurements are presented as mean±sem. *, P <0.05; **, P <0.005; ***, P <0.0005; ****, P <0.0001.
5 shows that both PS and TFPI are expressed in murine synovial fluid. A, Immunostaining for PS and TFPI in the intra-articular space of injured knees from F8 ^−/− Pros1 ^+/+ mice pretreated with control-siRNA or mPS-siRNA. Arrow heads point to synovial tissue and arrows to vasculature, all of which are positive for both PS and TFPI. Boxes in the upper figure (scale bar: 200 μm) show enlarged regions in the lower panel (scale bar: 50 μm). B, Immunostaining for TFPI in the intra-articular space of the uninjured knee of F8 ^-/- Pros1 ^+/+ and F8 - ^/- Pros1 ^-/- mice. Western blot analysis of conditioned media from primary murine fibroblast-like synovial cell (FLS) cultures using CE, anti-PS (c) and anti-TFPI (d) antibodies. Platelet-free plasma (PFP), protein lysate of platelets (PLT), and murine PS (mPS) were used as positive controls (c). TFPI isoform expression determined by comparing the molecular weight of deglycosylated and fully glycosylated TFPI. Murine placenta was used as a positive control for TFPIα. Western blot analysis of total protein lysates isolated from FLS after 24 hour incubation in the presence of thrombin (Thr, +) or vehicle (-) using EF, anti-PS (f) and anti-TFPI (e) antibodies. Human recombinant full-length TFPI was used as a positive control for TFPIα (hrTFPI). Blots are representative of 3 independent experiments.
6 shows PS and TFPI in human synovial fluid. A, PS and TFPI are expressed in synovial tissues of patients with HA (symptomatic and prophylactic), symptomatic HB or osteoarthritis (OA). Arrowheads point to synovial intimal layer and arrows to subintimal vascular structures, both of which are positive for both PS and TFPI. Scale bar: 50 μm. B, Western blot analysis of conditioned media of primary human FLS (hFLS) cultures from healthy individuals and OA patients before and after deglycosylation with anti-TFPI antibody. Human platelet lysate (hPLT) was used as a positive control for TFPIα. Blots are representative of 3 independent experiments.
Figure 7 shows thrombin generation and fibrin network in hemophilia A, TF-(1 pM) PRP from F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- mice showing TFPI-dependent PS activity. induced thrombin generation. B, APC-dependent PS activity in PRP and PFP from F8 ^{-/ -} Pros1 ^+/+ and F8 -/- Pros1 ^-/- mice. From C, F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- , and from F9 ^+/+ Pros1 ^+/+ , F9 ^-/- Pros1 ^+/+ and F9 ^-/- Pros1 ^-/- Representative scanning electron microscopy images from fibrin structures. DG, thrombin generation in patients with severe HA (FVIII <1%) in PFP (DE) and PRP (FG) without (D, F) and with (E, G) high titer inhibitors at low TF concentrations (1 pM). ) was triggered by Measurements are presented as mean±sem. **, P <0.005; ***, P <0.0005.
8 shows the genotyping approach. Genotypes obtained by mating F8 ^-/- Pros1 ^+/- (ac) and F9 ^-/- Pros1 ^+/- (df) mice. a, The Pros1 allele was amplified by multiplex PCR. PCR products were then subjected to electrophoresis; According to Saller, 2009, the wt band had a lower molecular weight (234 bp) compared to the null band (571 bp). b, Setup of multiplex PCR to amplify the wt band (620 bp) and null band (420 bp) of the F8 allele from genomic DNA. c, PCR product of F8 allele amplification ( null band: 420 bp) for the same sample as in (a). d, Pros1 alleles were amplified by multiplex PCR. PCR products were then subjected to electrophoresis; According to Saller, 2009, the wt band had a lower molecular weight (234 bp) compared to the null band (571 bp). e, Setup of multiplex PCR to amplify the wt band (320 bp) and null band (550 bp) of the F9 allele from genomic DNA. f, PCR product of F9 allele amplification ( null band: 550 bp) for the same sample as in (d).
9 shows histology in physiological condition. Insoluble in liver, lung, kidney, brain regions in F8 ^-/- Pros1 ^-/- and F8 ^{- /-} Pros1 ^{+/ +} mice and in F9 -/- Pros1 +/+ and F9 ^-/- Pros1 ^-/- Immunostaining method for fibrin. Scale bar: 100 μm.
10 shows that genetic loss of Pros1 prevents hematomas in hemophilia B mice. A, Difference between knee diameters 72 h post and pre-injury in F9 ^-/- Pros1 ^+/+ , F9 ^{- /-} Pros1 ^+/- , F9 -/- Pros1 ^-/- and F9 ^+/+ Pros1 ^+/+ mice. B, Microscopic evaluation of the intra-articular space of the knee of representative uninjured and injured limbs after 72 h in F9 ^+/+ Pros1 + / ⁺ , F9 ^-/ - Pros1 ^+/+ and F9 ^-/- Pros1 ^-/- mice (insoluble Mason's trichrome staining and staining for fibrin, mAb clone 102-10). Scale bar: 500 μm. Measurements are presented as mean±sem. ***, P <0.0005.
11 shows the quantification of fibrin network density and fiber branching. ab, Fibrin networks of F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- mice. cd, Fibrin networks of F9 ^+/+ Pros1 ^+/+ , F9 ^-/- Pros1 ^+/+ and F9 ^-/- Pros1 ^-/- . Quantification of fibrin net density (a and c). Quantification of fiber branching (b and d). Measurements are presented as mean±sem. ***, P <0.0005.

Example

Example 1: Loss of X-ase activity Pros1 ^-/- save the mouse

Pros1 ^+/- females mated with F8 ^-/- males produced 25% F8 ^+/- Pros1 ^+/- offspring. F8 ^+/- Pros1 ^+/- females bred with F8 ^- /- males produced 25% F8 ^-/- Pros1 ^+/- offspring (FIGS. 8A-C). Similar observations were made in F9 ^-/- Pros1 ^+/- mice (Fig. 8D-F). As expected, F8 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^-/- mice did not show FVIII and FIX plasma activity, respectively, and PS did not show F8 ^-/- Pros1 ^-/- and F9 ^-/- It was not detected in the plasma of Pros1 ^-/- mice (Fig. 1C-D). As reported ^, PS levels in F8 - ^{/- Pros1} +/- ^and F9 ^{- /-} Pros1 ^{+/- were} ~ 50- 60% lower (Fig. 1C–D).

Of the 295 pups from the F8 ^-/- Pros1 ^+/- breeding pair, 72 (24%) were F8 ^-/- Pros1 ^+/+ and 164 (56%) were F8 ^-/- Pros1 ^+/- and 59 animals (20%) were F8 ^-/- Pros1 ^-/- (χ ² =4.8, P =0.09). Therefore, F8 ^-/- Pros1 ^-/- mice were present at the expected Mendelian ratio. In contrast, of 219 pups from the F9 ^-/- Pros1 ^+/- breeding pair, 56 (26%) were F9 ^-/- Pros1 ^+/+ and 132 (60%) were F9 ^-/- Pros1 ^+/- and 31 (14%) were F9 ^-/- Pros1 ^-/- (χ ² =14.95, P =0.001). This is compatible with transmission ratio distortion for F9 ^-/- Pros1 ^-/- mice and consistent with reduced litter size compared to crosses from F9 ^+/+ Pros1 ^+/+ mice (5.2± 0.7 vs. 9.8±1.8, n = 4 matings/over 3 ^t generations, P = 0.046).

F8 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^-/- mice appeared completely normal. Their viability was monitored up to 20 (n=4) and 16 months (n=2), respectively, and showed no difference compared to F8 ^-/- Pros1 ^+/+ and F9 ^-/- Pros1 ^+/+ mice, respectively. .

Since complete Pros1 deficiency in mice leads to coagulopathy of wasting ¹⁵ , we evaluated whether F8 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^-/- mice developed DIC. DIC variables were found in F8 ^-/- Pros1 ^+/+ , F8 ^-/- Pros1 ^+/- and F8 ^-/- Pros1 ^-/- mice (Fig. 1C), and in F9 ^-/- Pros1 ^+/+ , F9 ^-/- Boiled in Pros1 ^+/- and F9 ^-/- Pros1 ^-/- mice (FIG. 1D). The activated fractional thromboplastin times are F8 ^-/- Pros1 ^+/+ (69±2 sec), F8 ^-/- Pros1 ^+/- (68±3 sec) and F8 ^-/- Pros1 ^- due to the absence of FVIII. ^/− (63±3 s) were equally prolonged in mice (mean±sem, n =6 per group, P =0.3). Boiling data were obtained using F9 ^-/- Pros1 ^+/+ , F9 ^-/- Pros1 ^+/- and F9 ^-/- Pros1 ^-/- mice. Moreover, no thrombosis or fibrin deposition was found in the brain, lung, liver and kidney of F8 ^-/- Pros1 ^-/- and F9 - ^/- Pros1 ^-/- mice (FIG. 9).

Thus, loss of X-ase activity rescues embryonic lethality of complete Pros1 deficiency. However, rescue was partial due to loss of FIX activity. A possible explanation is that severe HB appears to be a less severe condition than severe HA. Consequently, F9 disruption in Pros1 ^−/− mice was less efficient in coagulation readjustment than F8 disruption.

To investigate whether restoring native X-ase activity by FVIII infusion induces DIC, thrombosis and fulminant purpura in F8 ^-/- Pros1 ^-/- mice, recombinant FVIII (rFVIII) was administered intravenously. . No mice died after rFVIII injection. Numerous intravascular clots and hemorrhages in the lungs were found in F8 ^-/- Pros1 ^-/- mice 24 h after a single injection of excess rFVIII (FIG. 1E-F). Twenty-four hours after repeated administration of normal doses of rFVIII, coagulation assays showed non-coagulable prothrombin time (PT) (not shown), low fibrinogen, and high thrombin-antithrombin (TAT) levels, consistent with apparent DIC ( Figure 1G). In contrast, F8 ^-/- Pros1 ^-/- After a single injection of normal doses of rFVIII in mice, fibrinogen and TAT levels decreased in untreated F8 ^-/- Pros1 ^-/- Boiled with mice (Fig. 1G). Although numerous blood clots were seen in the lungs and liver (Fig. 1H-I), none of these mice developed purpura fulminosis.

Example 2: Loss of X-ase activity Pros1 ^-/- Does not prevent death by TF-induced thromboembolism in mice

We previously found that 88% of Pros1 ^+/+ mice survived a TF-induced thromboembolism model, but only 25% of Pros1 ^+/- mice were still alive 20 min after injection of a low TF dose (~1.1 nM) proved. When using higher TF doses (~4.3 nM), both Pros1 ^+/+ and Pros1 ^+/- mice died within 20 minutes. However, Pros1 ^+/− died earlier than Pros1 ^+/+ . HA and WT mice were equally sensitive to this high TF-dose, and more than 85% of them died within 15 minutes (FIG. 2A). In contrast, >75% WT mice survived under the prothrombotic strategy with low molecular weight heparin (LMWH) (Fig. 2A). Thus, unlike LMWH, HA does not protect mice from TF-induced thromboembolism. Then on the same model F8 ^-/- Pros1 ^+/+ , F8 ^-/- Pros1 ^+/- and F8 ^-/- Pros1 ^-/- mice were investigated. After injection of TF (~2.1 nM), 40–60% of mice died (P > 0.05) regardless of their Pros1 genotype (Fig. 2B). However, F8 ^-/- Pros1 ^-/- and F8 ^-/- Pros1 ^+/- died earlier than F8 ^-/- Pros1 ^+/+ mice, and F8 ^-/- Pros1 ^+/- mice died earlier than F8 ^-/- Pros1 ⁺ There was a tendency to die earlier than ^/+ mice (average time to death: F8 ^-/- Pros1 ^+/+ 12±4 min , 7±2 min for F8 ^-/- Pros1 ^+/- and 8±3 min for F8 ^-/- Pros1 ^-/- mice, n= 4-6/group, P = 0.43). F9 ^-/- Pros1 ^+/+ , F9 ^-/- Pros1 ^+/- and F9 ^-/- Pros1 ^-/- mice (data not shown).

Fibrin clots were detected in the pulmonary arteries of F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- mice that died during TF-induced thromboembolism challenge (FIG. 2C). Importantly, there were more blood clots in the lungs of F8 ^−/− Pros1 − ^/− than F8 ^−/− Pros1 ^+/+ mice (n = 48 versus 26, respectively). In addition, most arteries in the F8 ^−/− Pros1 ^−/− lung were completely occluded, whereas in the F8 ^−/− Pros1 ^+/+ lung only partially occluded.

None of the F8 ^-/- Pros1 ^-/- mice that died during TF-induced thromboembolism-challenge developed fulminant purpura. F9 ^-/- Pros1 ^+/+ , F9 ^-/- Pros1 ^+/- and F9 ^-/- Pros1 ^-/- mice (not shown).

Example 3: Loss of FVIII from thrombosis in mesenteric arterioles Pros1 ^-/- partially protects the mouse

Thrombus formation in mesenteric arterioles, a model sensitive to binding in the intrinsic coagulation pathway, was then recorded. In F8 ^+/+ Pros1 ^+/+ mice, thrombi grew to occluded size within 20 minutes, and all injured arteries were occluded (Fig. 2D). As expected, arterioles of F8 ^−/− Pros1 ^+/+ did not show thrombosis, whereas F8 ^−/− Pros1 ^−/− mice showed partial thrombosis ( FIG. 2D ).

Emboluses were formed during clot formation in F8 ^+/+ Pros1 ^+/+ mice but not in F8 ^-/- Pros1 ^+/+ mice. In F8 ^-/- Pros1 ^-/- mice, multiple micro-emboli were isolated during partial thrombus growth to prevent occlusive thrombus formation.

Example 4: in mice with HA Pros1 Targeting is limited, but tail hemorrhage is not eliminated

Bleeding phenotype was assessed by tail incision using mild or severe bleeding models.

In both models, blood loss was reduced in F8 ^−/− Pros1 ^−/− compared to F8 ^−/− Pros1 ^+/+ mice ( FIGS. 3A-B ). When challenged with the mild model, F8 ^−/− Pros1 ^+/− mice bleed less than F8 ^−/− Pros1 ^+/+ mice ( FIG. 3A ). In contrast, when exposed to the severe model, F8 ^-/- Pros1 ^-/- and F8 ^-/- Pros1 ^+/- mice exhibited comparable blood loss (Fig. 3B). However, F8 ^−/− Pros1 ^−/− mice bled more than F8 ^+/− Pros1 ^+/+ and F8 ^+/+ Pros1 ^+/+ mice in both models (Fig. 3A-B), indicating that F8 ⁻ Indicates that loss of Pros1 in ^/- mice only partially corrected the bleeding phenotype of F8 ^-/- mice.

We then investigated how inhibition of PS activity alters tail hemorrhage in F8 ^-/- Pros1 ^+/- mice using a PS-neutralizing antibody. This antibody limited blood loss in F8 ^−/− Pros1 ^+/− mice ( FIG. 3C ) to the same extent as complete genetic loss of Pros1 ( FIG. 3B ).

Example 5: Pros1 Targeting or PS inhibition completely protects HA or HB mice from acute hemorrhagic arthrosis (AH)

Bleeding can occur anywhere in hemophiliacs, but most bleeding occurs in the joints. To determine whether Pros1 loss prevents hemorrhagic arthrosis in hemophiliac mice, F8 ^-/- Pros1 ^+/+ , F8 ^-/- The AH model was applied to Pros1 ^+/- , F8 ^-/- Pros1 ^-/- and F8 ^+/+ Pros1 ^+/+ mice. Knee swelling after injury was reduced in F8 ^-/- Pros1 ^-/- and F8 ^+/+ Pros1 + ^/+ mice compared to F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^+/- mice (Fig. 4A ). There was no difference in knee swelling between F8 ^-/- Pros1 ^-/- and F8 ^+/+ Pros1 ^+/+ mice (FIG. 4A). Bleeding was observed in the joint space and synovial fluid of F8 ^-/- Pros1 ^+/+ (IBS=2, n = 5), but F8 ^-/- Pros1 ^-/- (IBS=0, n = 5) and F8 ^+/+ Pros1 It was not observed in ^+/+ mice (IBS=0, n =5) (Fig. 4B). There was more fibrin in the joint space and synovial fluid of F8 ^−/− Pros1 ^+/ + than F8 − ^/− Pros1 ^−/− and F8 ^+/+ Pros1 ^+/ + mice (Fig. 4B). F9 ^-/- Pros1 ^+/+ and F9 ^-/- Pros1 ^-/- Similar data were obtained for mice (IBS=0, n =3 and IBS=2, n =3, respectively) (FIGS. 9A-B).

These results were confirmed by continuous subcutaneous injection (starting 1 day before AH induction) of PS neutralizing antibody or control antibody in F8 ^-/- Pros1 ^+/- mice for 4 days (knee edema was 0.43±0.07 in PS-neutralizing antibody group). It was 0.69 ± 0.09 mm in the control group versus the control group, n = 9, P = 0.04). PS plasma levels were 26±6% in the PS-neutralizing antibody group versus 45±3% in the control ( n =5, P =0.017). In addition, PS inhibition was alternatively achieved by intravenous injection of murine PS (mPS) siRNA prior to AH challenge in F8 ^-/- Pros1 ^+/- and F8 ^-/- Pros1 ^+/+ mice (Fig. 4C-D) . IBS assessment showed no intra-articular hemorrhage in F8 ^-/- Pros1 ^+/+ mice treated with mPS siRNA (IBS = 0.5, n = 3) when compared to those treated with control siRNA (IBS = 2, n = 3) confirmed, (FIG. 4C). Importantly, PS expression was significantly reduced in plasma (26±3% vs control 84±11%, n =3, P =0.006) and synovial fluid were all reduced by mPS siRNA (Fig. 5A).

Example 6: Both PS and TFPI are expressed in the synovial fluid of mice

To understand the significant intra-articular hemostatic effect of genetic loss of Pros1 and PS suppression in hemophilia mice, knee sections were immunostained for PS and TFPI. PS was mainly present in the intimal layer of synovial tissues of F8 ^-/- Pros1 ^+/+ mice with AH treated with control siRNA, whereas synovial staining for PS was found in F8 ^-/- Pros1 ^+/+ mice with AH treated with mPS siRNA. ⁺ significantly reduced in mice (Fig. 5A). In contrast, TFPI staining was more pronounced in synovial tissue from hemophiliac mice receiving mPS siRNA than when treated with control siRNA (FIG. 5A). However, TFPI expression was reduced in F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- Boiled in the synovial lining layer of all mice (Fig. 5B).

To further demonstrate that PS is expressed by fibroblast-like synovial cells (FLS), collections from F8 ^+/+ Pros1 ^+/+ , F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- FLS Western blot was performed in conditioned medium. As shown in Figure 5C, the medium of F8 ^+/+ Pros1 ^+/+ and F8 ^-/- Pros1 ^+/+ FLS showed a band at a molecular weight of ~ 75 kDa, which was comparable to PS and observed in plasma and platelets. similar to what has been As expected, no staining was detected in media obtained from F8 ^+/+ Pros1 ^-/- FLS (FIG. 5C).

TFPI expression was also studied in F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- FLS conditioned media (FIG. 5D). All media showed a band at ~50 kDa, similar to that observed in placental lysates. TFPI isoform expression was investigated after protein deglycosylation, since fully glycosylated TFPIα and TFPIβ migrate at the same molecular weight ³⁰ . TFPI deglycosylated from the FLS medium migrated as a single band at a molecular weight of TFPIα similar to that of placental TFPI (positive control for TFPIα) (FIG. 5D). This indicates that FLS expresses TFPIα but not TFPIβ. Moreover, PS and TFPI expression increased in F8 ^−/− Pros1 ^+/+ FLSs after thrombin stimulation (Fig. 5E–F).

Example 7: Both PS and TFPI are expressed in the synovial fluid of HA or HB patients

Human HA, HB and osteoarthritic knee synovial tissues were then analyzed for both PS and TFPI (FIG. 6A). Strong signals for TFPI and PS were found in the synovial intimal and subintimal layers of allopathic HA patients (n = 7). In contrast, immunostaining for both PS and TFPI was reduced in HA patients on prophylaxis (n=5). Patients with symptomatic HB showed fewer signals for both PS and TFPI in the synovial intimal and subintimal layers ( n =4) than patients with symptomatic HA. Sections from osteoarthritis patients (n = 7) did not show strong staining for TFPI and PS, similar to hemophilia patients on prophylaxis. To assess which TFPI isoforms are expressed by human FLS, Western blots were performed on conditioned media of human FLS isolated from healthy subjects and osteoarthritis patients. Similar to murine FLS, human FLS express TFPIα but not TFPIβ (Fig. 6B).

Example 8: Pros1 Loss of is responsible for TFPI-dependent PS inactivation and resistance to APC in HA mice

Complete protection against AH in HA or HB mice lacking Pros1 or suppressed PS can be explained, at least in part, by the absence of PS cofactor activity for APC and TFPI in the joint. However, the reason for the partial hemostatic effect of no Pros1 or PS inhibition in HA mice challenged in the tail bleeding model needs to be further investigated.

An ex vivo TF-initiated thrombin generation test showed a correlation between the dose of thrombin-generating plasma and the clinical severity of hemophilia. Therefore, the effect of Pros1 loss on thrombin generation in the plasma of HA mice was investigated. TFPI-dependent PS activity was evaluated in platelet-rich plasma (PRP) but not in platelet-free plasma (PFP) because the TFPI-cofactor activity of PS could not be demonstrated in mouse plasma using the thrombin generation test. am. This is explained by the absence of TFPIα in mouse plasma and its presence in mouse platelets.

Thrombin peak and endogenous thrombin potential (ETP) were significantly higher in F8 ^-/- Pros1 ^-/- than in F8 ^-/- Pros1 ^+/+ PRP in response to 1 pM TF (1072±160 vs. 590±10 nmol/L.min, n =3/group, P =0.04), suggesting no PS TFPI-cofactor activity in F8 ^-/- Pros1 ^-/- PRP (Fig. 7A). Consistent with previous studies, thrombin peaks and ETP were increased by F8 ^−/− Pros1 ^+/+ in the presence of 1, 2.5 or 5 pM TF. and F8 ^-/- Pros1 ^-/- mice in PFP (data not shown).

To assess whether F8 ^-/- Pros1 ^-/- mice displayed defective functional APC-dependent PS activity, in the absence of APC, in the presence of wild-type (WT) recombinant APC, or mutated (L38D) recombinant mouse APC (L38D APC, a variant with deleted PS cofactor activity) was used to test for thrombin generation in Ca ²⁺ ionophore-activated PRP. In this assay, APC titration showed that the addition of 8 nM WT APC could reduce ETP by 90% in activated PRP of WT mice, whereas the same concentration of L38D APC reduced ETP by only 30% (data not shown). ) was shown. Based on these data, thrombin generation curves were recorded for activated PRP (3 mice/assay). The calculated APC ratio (ETP _{+ APC WT} / ETP _{+APC L38D} ) showed APC resistance in F8 ^-/- Pros1 ^-/- p plasma but not in F8 ^-/- Pros1 ^+/+ plasma (0.87±0.13 versus 0.87±0.13, respectively). 0.23±0.08, P =0.01) (Fig. 7B).

APC-dependent PS activity was also tested in PFP of F8 ^-/- Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- mice (2 mice/assay) in the presence of 2 nM WT APC and L38D APC. The calculated APC ratios showed APC resistance in F8 ^−/− Pros1 ^−/− but not F8 ^−/− Pros1 ^+/+ mice (1.08±0.04 versus 0.25±0.09, respectively, P=0.0003) ( FIG. 7B ).

Example 9: Pros1 Improved fibrin network in HA mice without

The tail-bleeding mouse model is sensitive to changes in coagulation and fibrinolysis as well as platelet dysfunction. To understand the differences between the genotypes studied with respect to tail hemorrhage, scanning electron microscopy images were used to examine the fibrin structure (Fig. 7C). Clots from F8 ^+/+ Pros1 ^+/+ and F8 ^-/- Pros1 ^-/- plasma showed a denser network of highly branched fibrin fibers compared to F8 ^-/- Pros1 ^+/+ plasma clots. (Fig. 11a-b). In contrast, clots from F9 ^+/+ Pros1 ^+/+ and F9 ^-/- Pros1 ^-/- plasma did not show a denser network than F9 ^-/- Pros1 ^+/+ plasma clots, but showed a tendency towards increased fiber branching. showed (Fig. 11c-d).

Fibrin fibers from F8 ^-/- Pros1 - ^{/ -} and F8 ^-/- Pros1 ^+/+ mice , and F9 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^{+/+ mice} were showed a larger diameter compared to fibers from either ^/+ mice or F9 ^+/+ Pros1 ^+/+ mice, respectively. Nevertheless, the fiber surfaces of F8 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^-/- mice were less porous compared to F8 ^-/- Pros1 ^+/+ or F9 ^-/- Pros1 ^+/+ mice, respectively. , which indicates that F8 ^-/- Pros1 ^-/- and F9 ^-/- Pros1 ^-/- -derived fibers are more permeable than F8 ^-/- Pros1 ^+/+ or F9 ^-/- Pros1 ^+/+ -derived fibers. It is smaller, suggesting that it may be more resistant to fibrinolysis ³⁸ . These data complement both the TFPI and APC cofactor activity results ( FIGS. 7A-B ), suggesting that tail hemorrhage in F8 ^−/− Pros1 ^−/− was improved compared to F8 −/− Pros1 +/+ mice, but not F8 ^−/− Pros1 ^+/+ mice. ^+/+ Pros1 ^+/+ helps explain why it is not completely corrected as in the mouse.

Example 10: PS Inhibition in Plasma Restores Thrombin Generation in HA Patients

We then investigated the effect of PS inhibition on thrombin generation in human HA plasma. The ETP of PFP increased 2-4 fold in the presence of PS-neutralizing antibodies. Similar results for efficient FXa inhibition were obtained using an anti-human TFPI antibody directed against the C-terminal domain even in the presence of a FVIII inhibitor (FIGS. 7D-E). PS inhibition had a significant effect when ETP increased more than 10-fold in PRP samples (1912±37 and 1872±64 nM*min) (Fig. 7F and G, respectively). Thus, PS inhibition completely restored ETP in hemophiliac plasma (for comparison, ETP in normal plasma: 1495±2 nM*min). Similar results were obtained using an anti-TFPI antibody (Fig. 7D-G). These data confirm in humans the improvement in thrombin generation in HA PFP and PRP induced by PS inhibition observed in mice.

Example 11: Materials and Methods

mouse

F8 ^-/- mice ( B6;129S4- F8 ^tm1Kaz / J ) and F9 ^-/- mice ( B6.129P2- F9 ^tm1Dws / J ) from a C57BL/6J background were obtained from The Jackson Laboratory . Pros1 ^+/- Mice were progeny of the original colony ¹⁵ . The Swiss Federal Association of Veterinarians approved the experiment. Mice were genotyped as described in the literature ^15-17 .

TF-induced pulmonary embolism

The venous thromboembolic model was adapted from Weiss et al. ¹⁸ with minor modifications ¹⁵ . Anesthetized mice aged 6-9 weeks received intravenous (2 μL/g) administration of 4.25 nM (1:2 dilution) or 2.1 nM (1:4 dilution) of human recombinant TF (hrTF, Dade Innovin, Siemens) . Lungs were harvested 2 min after the onset of respiratory arrest or at the end of the 20 min observation period and fixed in 4% PFA. Lung sections were stained with hematoxylin and eosin and for fibrin. The extent of fibrin clots in the lungs was evaluated as the number of intravascular thrombi in 10 randomly selected non-overlapping areas (×10 magnification).

Tail clipping model in HA mice

Two different tail clipping models for assessing the bleeding phenotype were evaluated as described ¹⁴ . Briefly, the distal tail of 8–10 week old mice was transected at 2 mm (mild injury), bleeding was venous or 4 mm (severe injury), and bleeding was arterial and venous ¹⁹ . Bleeding was quantified as blood lost after 30 or 10 minutes, respectively. In severe injury models, some F8 ^-/- Pros1 ^+/- Mice received an intravenous dose of rabbit anti-human PS-IgG (Dako) or rabbit isotype IgG (R&D Systems) at a dose of 2.1 mg/kg 2 minutes before tail transection.

Acute hemorrhagic arthrosis model

According to the literature of Øvlisen et al ^{. 20} , induction of joint hemorrhage, measurement of knee diameter, and analgesia coverage were performed in anesthetized 9-12 week old mice. Joint diameter was measured with a digital caliper (Mitutoyo 547-301, Kanagawa) at 0 and 72 h. At 72h, mice were sacrificed and knees were isolated, fixed in 4% PFA, decalcified and immersed in paraffin. Intra-articular bleeding score (IBS) was assessed as described in the literature ²¹ .

PS inhibition in vivo

10-week-old mice were continuously infused with rabbit anti-human PS-IgG (Dako Basel, Switzerland) or rabbit isotype IgG (R&D Systems) at 1 mg/kg/day via subcutaneous osmotic minipump (model2001, Alzet).

Alternatively, 10-week-old mice were transfected with a single dose of 1 mg/kg mouse-specific siRNA (s72206, Life Technologies) or control siRNA (4459405) using transfection material (Invivofectamine 3.0, Invitrogen, Life Technologies) according to the manufacturer's instructions. , In vivo negative control #1 Ambion, Life Technologies). An acute hemorrhagic arthrosis model was applied 2.5 days after PS inhibition.

statistical method

Values were expressed as mean ± sem. Mendelian allelic segregation was assessed using chi-square for non-linked genetic loci. Survival data in the TF-induced venous thromboembolic model were plotted using the Kaplan-Meier method. Curves were statistically compared using the log-rank test (Prism 6.0d; GraphPad). Other data were analyzed by t-test, one-way and two-way ANOVA tests using GraphPad Prism 6.0d. A P -value of less than 0.05 was considered statistically significant.

Preparation of murine plasma

6-9 week old mice were anesthetized with pentobarbital (40 mg/kg), and whole blood was drawn into 3.13% citrate from the inferior vena cava (1 vol anticoagulant/9 vol blood). Blood was centrifuged at 1031 g for 10 min in a centrifuge preheated to 26 °C to obtain platelet-rich plasma (PRP). Alternatively, blood was centrifuged at room temperature (RT) at 2400g for 10 minutes to obtain platelet-poor plasma (PPP). To obtain platelet-free plasma (PFP), additional centrifugation was performed at 10000g for 10 minutes.

Measurement of platelet count and coagulation parameters

Platelet counts were performed with an automatic cell counter (Procyte Dx Hematology Analyzer, IDEXX). Fibrinogen, FVIII and FIX activities were measured on an automated Sysmex CA-7000 coagulation analyzer (Sysmex Digitana). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured in a coagulometer (MC4plus, Merlin Medical).

Measurement of murine PS antigen and TAT complexes by ELISA

Wells of a 96-well plate (Maxisorb, Thermo) were coated with 50 μL per well of 10 μg/mL rabbit polyclonal anti-human PS (DAKO Cytomation) and incubated overnight at 4°C. After washing three times with TBS buffer (0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 7.5, 0.05% Tween 20), the plate was blocked with 2% TBS-BSA. Diluted plasma samples (dilution range: 1:300-1:600) were added to the wells and incubated for 2h at RT. After washing three times, 50 μL of 1 μg/mL biotinylated chicken polyclonal anti-murine protein S was added and incubated for 2 hours at room temperature. Streptavidin-HRP conjugated horseradish peroxidase (Thermo) was added to amplify the signal and the plate was incubated for 1 hour. The plate was washed 3 times and 100 μLTMB substrate (KPL) was added. The reaction was stopped by adding 100 μL HCl (1M). Absorbance was measured at 450 nm. A standard curve was established using serial dilutions of pooled normal plasma from 14 healthy mice (8 males and 6 females, 7-12 weeks old). Results for pooled normal plasma are expressed as percentages.

TAT levels were measured in duplicate for each plasma sample using a commercial ELISA (Enzygnost TAT micro, Siemens) according to the manufacturer's instructions.

Mouse tissue processing and sectioning, immunochemistry and microscopy

Untreated tissue sections (4 μm) were stained with hematoxylin/eosin or Mason's trichrome or immunostained for insoluble fibrin, PS or TFPI. The following antibodies were used: fibrin (mAb clone 102-10) ¹ final concentration 15.6 μg/mL, incubated for 30 min at RT, secondary antibody rabbit anti-human, (ab7155 Abcam, Cambridge, UK) 1:200 dilution, 30 Incubate at RT for min; PS (MAB 4976, R&D, dilution 1:50) incubate at RT for 30 min, secondary antibody rabbit anti-rat, (ab7155 Abcam) - 1:200 dilution, incubate at RT for 30 min; TFPI (PAHTFPI-S, Hematological Technologies) final concentration 18.6 μg/mL, incubated at RT for 30 min, secondary antibody rabbit anti-sheep IgG (ab7106, Abcam) 1:200 dilution, incubated at RT for 30 min. All staining was performed with the immunostaining agent BOND RX (Leica Biosystems, Muttenz, Switzerland) according to the manufacturer's instructions. Entire slides were scanned using a 3D HISTECH Panoramic 250 Flash II with 20x (NA 0.8), 40x (NA 0.95) air objectives. Image processing was performed using Panorama Viewer software.

In vivo administration of FVIII to mice with complete loss of F8

6-9 week old mice were anesthetized with ketamine (80mg/kg) and xylazine (16mg/kg). 0.3 U/kg of recombinant FVIII (Advate® Baxalta) at 1 h until 100% FVIII level is reached (normal dose) or excess recombinant FVIII (2 U/kg) at 1 h until >200% FVIII level is reached administered intravenously. Normal dose or overdose was injected 1 h before and 1 h after the introduction of the jugular catheter (mouse JVC 2Fr PU 10 cm, Instech) and 4 h, 8 h and 16 h after midline placement. Mice were sacrificed 24 h after the first injection. Blood was drawn and organs were harvested. FVIII, fibrinogen and thrombin-antithrombin complex (TAT) were measured as described in the Examples. Lungs were isolated, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin.

FeCl in the mesenteric artery ₃ Injury thrombosis model

A mesenteric artery thrombosis model was performed using biomicroscopy according to Ref. ² but with minor modifications. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (16 mg/kg). Platelets were directly labeled in vivo by injection of 100 μL Rhodamine 6G (1.0 mM). After selection of study area, vessel wall damage was created by topical treatment for 1 min with a filter paper (1 mm diameter 1M Whatmann paper patch) saturated with 10% FeCl ₃ . Thrombus formation was monitored in real time under a fluorescence microscope (IV-500, Micron instrument, San Diego, Calif.) equipped with a FITC filter set equipped with affinity-corrected immersion optics (Zeiss, Germany). Brightly fluorescently labeled platelets and leukocytes were observed in a 1355 μm X 965 μm field of view via video-triggered stroboscopic epi-illumination (Chadwick Helmuth, El Monte, CA). A 10X objective lens of NA0.3. Zeiss Plan-Neofluar was used. All scenes were recorded to videotape using a custom low-latency silicon-enhanced target camera (Dage MTI, Michigan city, IN), a timebase generator, and a Hi-8 VCR (EV, C-100, Sony, Japan). The vessel wall occlusion time, determined by cessation of blood cell flow, was measured.

Fibroblast-Like Synovial Cell (FLS) Isolation, Culture and Flow Cytometry

Murine FLS were isolated and cultured from 8-10 week old mice according to literature ³ . After 3 passages, phase-contrast images of cells were taken, and cells were incubated with FITC-conjugated rat anti-mouse CD11b antibody (M1/70, Pharmingen, BD Biosciences), PE-conjugated rat anti-mouse CD90.2 antibody (30-H12 , Pharmingen, BD Biosciences), FITC-conjugated rat anti-mouse CD106 antibody (429 MVCAM.A, Pharmingen, BD Biosciences), PE-conjugated hamster anti-mouse CD54 antibody (3E2, Pharmingen, BD Biosciences) and fluoro Incubation with chromium-conjugated isotype control antibody for 30 minutes at 4°C in the dark. After final washing and centrifugation steps, all cultured cells were analyzed on an LSR II flow cytometer (BD Biosciences) and FACS Diva 7.0 software (BD Biosciences). Human FLS from healthy individuals and OA patients were purchased from Asterand, Bioscience and cultured according to the manufacturer's instructions.

western blotting

PS and TFPI were detected in human and mouse samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gradient SDS-PAGE, Bio-Rad) under reducing conditions. After protein transfer to a nitrocellulose membrane (Bio-Rad), murine PS was visualized using 2 μg/mL monoclonal MAB-4976 (R&D Systems) and 1 μg/mL polyclonal AF2975 (R&D Systems) for murine TFPI. Recombinant murine PS ⁴ (30 ng), recombinant human TFPI full length (provided by T. Hamuro, Kaketsuken, Japan), lysate of washed platelets, PFP from F8 ^-/- Pros1 ^+/+ mice and F8 ^+/+ Pros1 ^{+/ +} Placental lysates from mice were used as PS and TFPIα controls. Samples from confluent murine and human FLS conditioned medium were collected after 24h-incubation in serum-free medium (OptiMem) and concentrated 40-fold using an Amicon filter device (Millipore, 10 kDa cutoff). For TFPI Western blotting, samples were incubated with five protein deglycosylase (PNGase F, O-glycosidase, neuramidase, β-galactosidase, β-acetylglucosaminases) for 12 h at 37 °C before loading on the gel. Nidase, Deglycosylation Kit, V4931, Promega). Final detection was completed using horseradish peroxidase-conjugated secondary antibody (Dako) and Supersignal West Dura long term chemiluminescent substrate (Pierce) and monitored using a Fuji LAS 3000IR CCD camera.

Immunohistochemistry of human knee synovial fluid

Paraffin-embedded specimens of synovial tissue from 12 HA patients and 4 HB patients who underwent arthroplasty for severe knee arthrosis were collected from the Department of Laboratory and Clinical Medicine, Department of Anatomy and Histology, University of Florence, as described elsewhere ^5,6 . Collected from the archives. Seven HA patients were treated as needed and five were treated with secondary prophylaxis. All 4 HB patients were treated as needed. Synovial fluid samples from 7 osteoarthritis (OA) patients were used as controls ^5,6 . For immunohistochemical analysis, synovial tissue sections (5 μm thick) were deparaffinized, rehydrated, and boiled for 10 min in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval followed by 3% in methanol. Endogenous peroxidase activity was blocked by treatment with H ₂ O ₂ at room temperature for 15 minutes. The slices were then washed in PBS and incubated with Ultra V block (UltraVision High Volume Detection System Anti-Polyvalent, HRP, catalog number TP-125-HL, LabVision) for 10 minutes at room temperature according to the manufacturer's protocol. After blocking non-specific site binding, slides were inoculated with rabbit polyclonal anti-human protein S/PROS1 antibody diluted in PBS (1:50 dilution, catalog number NBP1-87218, Novus Biologicals) or sheep polyclonal anti-human tissue factor pathway. inhibitor (TFPI) antibody (1:500 dilution, catalog number PAHTFPI-S, Haematologic Technologies) and incubated overnight at 4°C. For PS immunostaining, tissue sections were incubated with a biotinylated secondary antibody followed by streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) according to the manufacturer's protocol. For TFPI immunostaining, tissue sections were instead incubated with HRP-conjugated donkey anti-sheep IgG (1:1000 dilution; catalog number ab97125; Abcam) for 30 minutes. Immunoreactivity was developed using 3-amino-9-ethylcarbazole (AEC kit, catalog number TA-125-SA; LabVision) as a chromosome. Synovial sections were finally stained with Mayer's hematoxylin (Bio-Optica), washed, embedded in an aqueous loading medium, and observed under a Leica DM4000 B microscope (Leica Microsystems). Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune IgG (Sigma-Aldrich) were included as negative controls for antibody specificity. Optical microscope images were captured with a Leica DFC310 FX 1.4-megapixel digital color camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).

Investigation of fibrin clot ultrastructure

Fibrin clots were prepared at 37°C from PFP by adding ~5 nM TF (Dade Innovin, Siemens). They were then fixed in 2% glutaraldehyde, dehydrated, dried and sputter-coated with gold palladium for visualization using scanning electron microscopy according to Zubairova et al. ⁷ . Semi-quantitative evaluation of network density and fiber branching was performed using STEPanizer software (www.stepanizer.com).

Automated clot assay calibrated in murine samples

Thrombin generation in PFP and PRP was determined using a calibrated automated thrombogram (CAT) method.

TFPI-dependent PS activity was evaluated in PRP (150 G/L) as follows. Briefly, 10 μL mouse PRP (150 G/L) was mixed with 10 μL PRP reagent (Diagnostica Stago) and 30 μL of Buffer A (25 mm Hepes, 175 mm NaCl, pH 7.4, 5 mg/mL BSA). Thrombin generation was initiated at 37° C. using 10 μL of a fluorogenic substrate/CaCl ₂ mixture. The final concentrations were: 16.6% mouse plasma, 1 pM hrTF, 4 μM phospholipids, 16 mM CaCl ₂ , and 0.42 mM fluorogenic substrate.

APC-dependent PS activity was evaluated in a CAT-based APC resistance test of mouse PFP and PRP according to Dargaud Y et al. ⁸ . PRP (150 G/L) was pre-activated using 40 μM Ca ²⁺ ionophore (A23187) for 5 min at 37°C. Final concentrations were: 16.6% mouse plasma, 22 μM A23187, 1 pM hrTF, 4 μM phospholipids, 2 nM (for PFP) or 8 nM (for PRP) wild-type recombinant mouse APC (wt-rmAPC) ⁵ or mutant recombinant mouse APC (rmAPC L38D), 16 mM CaCl ₂ , and 0.42 mM fluorogenic substrate. Generation and characterization of rmAPC L38D was performed according to Refs ^9,10 and purification according to Refs ^11,12 .

For titration of TF to PFP, the following reagents were used: PPP reagent and MP reagent (Diagnostica Stago).

Fluorescence was measured using a Fluoroscan Ascent® fluorometer equipped with a dispenser. Fluorescence intensities were detected at wavelengths of 390 nm (excitation filter) and 460 nm (emission filter). The dedicated software program Thrombinoscope® version 3.0.0.29 (Thrombinoscope bv) was able to calculate the thrombin activity for the calibrator (Thrombinoscope bv) and displayed the thrombin activity over time. All experiments were performed twice at 37°C and measurements typically lasted 60 minutes.

CAT analysis in human samples

Written informed consent was obtained from the patients. Venous blood was collected by venipuncture in 3.2% sodium citrate (vol/vol) and centrifuged at 2000 g for 5 min. The platelet poor plasma (PPP) was then centrifuged at 10000 g for 10 minutes to obtain PFP. PFP was aliquoted, flash frozen and stored at -80 °C until use. For PRP, blood was centrifuged at 180 g×10 min. All subjects gave informed consent for participation. With minor modifications from reference ¹³ , thrombin generation was evaluated in human PFP and PRP. Briefly, 68 μL PFP or PRP (150 G/L) was added with 12 μL of polyclonal rabbit anti-human PS-IgG antibody (0.42 mg/mL, Dako) or monoclonal antibody to TFPI (0.66 μm, MW1848, Sanquin). or incubated with buffer A at 37 °C for 15 min. PFP samples were initiated with a 7:1 mixture of PPP low and PPP 5 pm reagents (Diagnostica Stago) or PRP samples were solidified with 20 μL of PRP reagent (Diagnostica stago). The addition of 20 μL of CaCl ₂ and a fluorogenic substrate (I-1140; Bachem) was followed by thrombin generation in a Fluoroskan Ascent reader (Thermo Labsystems).

Argument

As PS is a key regulator of thrombin generation, we envisioned that targeting PS could constitute a potential hemophilia therapy.

Extensive studies in mice provide proof-of-concept data supporting a pivotal role for PS and TFPI as a cause for hemorrhage and severe joint damage in hemophilic mice. Targeting Pros1 or inhibiting PS has the ability to ameliorate hemophilia in mice as judged by in vivo improvement of the bleeding phenotype and complete protection against hemorrhagic arthrosis in the tail bleeding assay (Figures 3A-C and 4). Because joints show very weak expression of TF and synovial cells produce large amounts of TFPIα and PS (FIG. 5), the activity of the extrinsic pathway is greatly reduced within the joint, making hemophilic joints prone to hemorrhage. Moreover, thrombomodulin (TM) and endothelial protein C receptor (EPCR) are expressed by FLS, which TM-thrombin complex activates EPCR bound-PC to produce APC, a very potent anticoagulant with respect to AH suggests Importantly, expression of TFPIα is upregulated by thrombin (FIG. 5F). Therefore, AH, which usually causes marked local inflammatory and joint symptoms that can last for days to weeks, also promotes the local production and secretion of a number of anticoagulants, namely APC, TFPIα, and their cofactor PS; This may help explain the pathophysiology of joint damage in hemophilia.

Observations using clinical samples from patients with hemophilia are consistent with those obtained from murine studies. In humans, blocking PS in the plasma of HA patients with or without inhibitors normalizes ETP (Fig. 7D-G). HB patients have less intraarticular expression of TFPI and PS than HA patients, consistent with current knowledge that HB patients have less bleeding than HA patients ( FIG. 6 ). Moreover, HA patients receiving prophylaxis showed less TFPI and PS synovial fluid expression than patients receiving FVIII concentrate only in a hemorrhagic situation, ie, so-called “symptomatic therapy” ( FIG. 6A ). Finally, human FLSs secrete both TFPIα and PS as observed in mice, making the extrapolation of murine hemophilia data to humans robust.

Based on the important findings in this report, we suggest that targeting PS could potentially translate into useful therapies for hemophilia. PS in the human and murine joints is a novel pathophysiological causative factor for hemorrhagic arthrosis and constitutes an attractive potential therapeutic target, particularly due to its dual cofactor activity on both APC and TFPIα in the joint. In the presence of PS, hemorrhagic arthritis increases TFPIα expression in synovial fluid. Targeting PS in mice protects mice from hemorrhagic arthrosis. Thus, we propose that both TFPIα and its cofactor PS produced by FLS, together with the TM-EPCR-PC pathway, comprise a potent intra-articular anticoagulant system with important pathological implications for hemorrhagic arthrosis. Murine PS silencing RNA (Fig. 4H-I and Fig. 5A), which we successfully used in hemophiliac mice, is a therapeutic approach that we will develop for hemophiliac patients. The advantage of silencing RNA over current factor replacement therapy is a longer half-life that reduces injection frequency and possible subcutaneous route of administration.

references

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2. Angelillo-Scherrer A, Fontana P, Burnier L, et al. Connexin 37 limits thrombus propensity by downregulating platelet reactivity. Circulation. 2011;124(8):930-939.

3. Armaka M, Vassiliki G, Kontoyiannis D, Kollias G. A standardized protocol for the isolation and culture of normal and arthritogenic murine synovial fibroblasts Protocol Exchange. 2009.

4. Fernandez JA, Heeb MJ, Xu X, Singh I, Zlokovic BV, Griffin JH. Species-specific anticoagulant and mitogenic activities of murine protein S. Haematologica. 2009;94(12):1721-1731.

5. Melchiorre D, Linari S, Manetti M, et al. Clinical, instrumental, serological and histological findings suggest that hemophilia B may be less severe than hemophilia A. Haematologica. 2016;101(2):219-225.

6. Melchiorre D, Milia AF, Linari S, et al. RANK-RANKL-OPG in hemophilic arthropathy: from clinical and imaging diagnosis to histopathology. J Rheumatol. 2012;39(8):1678-1686.

7. Zubairova LD, Nabiullina RM, Nagaswami C, et al. Circulating Microparticles Alter Formation, Structure, and Properties of Fibrin Clots. Sci Rep. 2015;5:17611.

8. Dargaud Y, Luddington R, Gray E, et al. Standardization of thrombin generation test--which reference plasma for TGT? An international multicentre study. Thromb Res. 2010;125(4):353-356.

9. Harmon S, Preston RJ, Ni Ainle F, et al. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement. J Biol Chem. 2008;283(45):30531-30539.

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18. Weiss EJ, Hamilton JR, Lease KE, Coughlin SR. Protection against thrombosis in mice lacking PAR3. Blood. 2002;100(9):3240-3244.

SEQUENCE LISTING <110> Universit? Bern <120> Use of siRNA against Protein S for the treatment of hemophilia <130> uz333wo <160> 68 <170> PatentIn version 3.5 <210> 1 <211> 3595 <212> DNA <213> Homo sapiens <400> 1 tttggaaacg tcacactgtg gaggaaaagc agcaactagg gagctggtga agaaggatgt 60 ctcagcagtg tttactaggc ctccaacact agagcccatc ccccagctcc gaaaagcttc 120 ctggaaatgt ccttgttatc acttcccctc tcgggctggg cgctgggagc gggcggtctc 180 ctccgccccc ggctgttccg ccgaggctcg ctgggtcgct ggcgccgccg cgcagcacgg 240 ctcagaccga ggcgcacagg ctcgcagctc cgcggcgcct agcgctccgg tccccgccgc 300 gacgcgccac cgtccctgcc ggcgcctccg cgcgcttcga aatgagggtc ctgggtgggc 360 gctgcggggc gctgctggcg tgtctcctcc tagtgcttcc cgtctcagag gcaaactttt 420 tgtcaaagca acaggcttca caagtcctgg ttaggaagcg tcgtgcaaat tctttacttg 480 aagaaaccaa acagggtaat cttgaaagag aatgcatcga agaactgtgc aataaagaag 540 aagccaggga ggtctttgaa aatgacccgg aaacggatta tttttatcca aaatacttag 600 tttgtcttcg ctcttttcaa actgggttat tcactgctgc acgtcagtca actaatgctt 660 atcctgacct aagaagctgt gtcaatgcca ttccagacca gtgtagt cct ctgccatgca 720 atgaagatgg atatatgagc tgcaaagatg gaaaagcttc ttttacttgc acttgtaaac 780 caggttggca aggagaaaag tgtgaatttg acataaatga atgcaaagat ccctcaaata 840 taaatggagg ttgcagtcaa atttgtgata atacacctgg aagttaccac tgttcctgta 900 aaaatggttt tgttatgctt tcaaataaga aagattgtaa agatgtggat gaatgctctt 960 tgaagccaag catttgtggc acagctgtgt gcaagaacat cccaggagat tttgaatgtg 1020 aatgccccga aggctacaga tataatctca aatcaaagtc ttgtgaagat atagatgaat 1080 gctctgagaa catgtgtgct cagctttgtg tcaattaccc tggaggttac acttgctatt 1140 gtgatgggaa gaaaggattc aaacttgccc aagatcagaa gagttgtgag gttgtttcag 1200 tgtgccttcc cttgaacctt gacacaaagt atgaattact ttacttggcg gagcagtttg 1260 caggggttgt tttatattta aaatttcgtt tgccagaaat cagcagattt tcagcagaat 1320 ttgatttccg gacatatgat tcagaaggcg tgatactgta cgcagaatct atcgatcact 1380 cagcgtggct cctgattgca cttcgtggtg gaaagattga agttcagctt aagaatgaac 1440 atacatccaa aatcacaact ggaggtgatg ttattaataa tggtctatgg aatatggtgt 1500 ctgtggaaga attagaacat agtattagca ttaaaatagc taaagaagct gtgatgg 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ctatgaa aaccagaaca aattttaaca aaaggacaac cacagaggga tatagtgaat 3300 atcgtatcat tgtaatcaaa gaagtaagga ggtaagattg ccacgtgcct gctggtactg 3360 tgatgcattt caagtggcag ttttatcacg tttgaatcta ccattcatag ccagatgtgt 3420 atcagatgtt tcactgacag tttttaacaa taaattcttt tcactgtatt ttatatcact 3480 tataataaat cggtgtataa ttttaaaatg catgtgaata tctttattat atcaactgtt 3540 tgaataaaac aaaattacat aatagacatt taactcttca aaaaaaaaaa aaaaa 3595 <210> 2 <211> 3682 <212> DNA <213> Homo sapiens <400> 2 gtcacactgt ggaggaaaag cagcaactag ggagctggtg aagaaggatg tctcagcagt 60 gtttactagg cctccaacac tagagcccat cccccagctc cgaaaagctt cctggaaatg 120 tccttgttat cacttcccct ctcgggctgg gcgctgggag cgggcggtct cctccgcccc 180 cggctgttcc gccgaggctc gctgggtcgc tggcgccgcc gcgcagcacg gctcagaccg 240 aggcgcacag gctcgcagct ccgcggcgcc tagcgctccg gtccccgccg cgacgcgcca 300 ccgtccctgc cggcgcctcc gcgcgcttcg aaatgagggt cctgggtggg cgctgcgggg 360 cgctgctggc gtgtctcctc ctagtgcttc ccgtctcaga ggcaaacttt tgtttatatt 420 ttagaaatga ttttatatac aaccgtgcat gcat 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ac acaaagtatg 1320 aattacttta cttggcggag cagtttgcag gggttgtttt atatttaaaa tttcgtttgc 1380 cagaaatcag cagattttca gcagaatttg atttccggac atatgattca gaaggcgtga 1440 tactgtacgc agaatctatc gatcactcag cgtggctcct gattgcactt cgtggtggaa 1500 agattgaagt tcagcttaag aatgaacata catccaaaat cacaactgga ggtgatgtta 1560 ttaataatgg tctatggaat atggtgtctg tggaagaatt agaacatagt attagcatta 1620 aaatagctaa agaagctgtg atggatataa ataaacctgg accccttttt aagccggaaa 1680 atggattgct ggaaaccaaa gtatactttg caggattccc tcggaaagtg gaaagtgaac 1740 tcattaaacc gattaaccct cgtctagatg gatgtatacg aagctggaat ttgatgaagc 1800 aaggagcttc tggaataaag gaaattattc aagaaaaaca aaataagcat tgcctggtta 1860 ctgtggagaa gggctcctac tatcctggtt ctggaattgc tcaatttcac atagattata 1920 ataatgtatc cagtgctgag ggttggcatg taaatgtgac cttgaatatt cgtccatcca 1980 cgggcactgg tgttatgctt gccttggttt ctggtaacaa cacagtgccc tttgctgtgt 2040 ccttggtgga ctccacctct gaaaaatcac aggatattct gttatctgtt gaaaatactg 2100 taatatatcg gatacaggcc ctaagtctat gttccgatca acaatctcat ctg gaattta 2160 gagtcaacag aaacaatctg gagttgtcga caccacttaa aatagaaacc atctcccatg 2220 aagaccttca aagacaactt gccgtcttgg acaaagcaat gaaagcaaaa gtggccacat 2280 acctgggtgg ccttccagat gttccattca gtgccacacc agtgaatgcc ttttataatg 2340 gctgcatgga agtgaatatt aatggtgtac agttggatct ggatgaagcc atttctaaac 2400 ataatgatat tagagctcac tcatgtccat cagtttggaa aaagacaaag aattcttaag 2460 gcatcttttc tctgcttata ataccttttc cttgtgtgta attatactta tgtttcaata 2520 acagctgaag ggttttattt acaatgtgca gtctttgatt attttgtggt cctttcctgg 2580 gatttttaaa aggtcctttg tcaaggaaaa aaattctgtt gtgatataaa tcacagtaaa 2640 gaaattctta cttctcttgc tatctaagaa tagtgaaaaa taacaatttt aaatttgaat 2700 ttttttccta caaatgacag tttcaatttt tgtttgtaaa actaaatttt aattttatca 2760 tcatgaacta gtgtctaaat acctatgttt ttttcagaaa gcaaggaagt aaactcaaac 2820 aaaagtgcgt gtaattaaat actattaatc ataggcagat actattttgt ttatgttttt 2880 gtttttttcc tgatgaaggc agaagagatg gtggtctatt aaatatgaat tgaatggagg 2940 gtcctaatgc cttatttcaa aacaattcct cagggggaac agctttggct tcatctttc t 3000 cttgtgtggc ttcacattta aaccagtatc tttattgaat tagaaaacaa gtgggacata 3060 ttttcctgag agcagcacag gaatcttctt cttggcagct gcagtctgtc aggatgagat 3120 atcagattag gttggatagg tggggaaatc tgaagtgggt acatttttta aattttgctg 3180 tgtgggtcac acaaggtcta cattacaaaa gacagaattc agggatggaa aggagaatga 3240 acaaatgtgg gagttcatag ttttccttga atccaacttt taattaccag agtaagttgc 3300 caaaatgtga ttgttgaagt acaaaaggaa ctatgaaaac cagaacaaat tttaacaaaa 3360 ggacaaccac agagggatat agtgaatatc gtatcattgt aatcaaagaa gtaaggaggt 3420 aagattgcca cgtgcctgct ggtactgtga tgcatttcaa gtggcagttt tatcacgttt 3480 gaatctacca ttcatagcca gatgtgtatc agatgtttca ctgacagttt ttaacaataa 3540 attcttttca ctgtatttta tatcacttat aataaatcgg tgtataattt taaaatgcat 3600 gtgaatatct ttattatatc aactgtttga ataaaacaaa attacataat agacatttaa 3660 ctcttcaaaa aaaaaaaaaa aa 3682 <210> 3 <211> 417 <212> DNA <213> Homo sapiens <400> 3 tttggaaacg tcacactgtg gaggaaaagc agcaactagg gagctggtga agaaggatgt 60 ctcagcagtg tttactaggc ctccaacact agagcccatc ccccagctcc gaa aagcttc 120 ctggaaatgt ccttgttatc acttcccctc tcgggctggg cgctgggagc gggcggtctc 180 ctccgccccc ggctgttccg ccgaggctcg ctgggtcgct ggcgccgccg cgcagcacgg 240 ctcagaccga ggcgcacagg ctcgcagctc cgcggcgcct agcgctccgg tccccgccgc 300 gacgcgccac cgtccctgcc ggcgcctccg cgcgcttcga aatgagggtc ctgggtgggc 360 gctgcggggc gctgctggcg tgtctcctcc tagtgcttcc cgtctcagag gcaaact 417 <210> 4 <211> 158 <212> DNA < 213> Homo sapiens <400> 4 ttttgtcaaa gcaacaggct tcacaagtcc tggttaggaa gcgtcgtgca aattctttac 60 ttgaagaaac caaacagggt aatcttgaaa gagaatgcat cgaagaactg tgcaataaag 120 aagaagccag ggaggtcttt gaaaatgacc cggaaacg 158 <210> 5 <211> 25 <212> DNA <213> Homo sapiens <400> 5 gattattttt atccaaaata cttag 25 <210> 6 <211> 87 <212> DNA <213> Homo sapiens <400> 6 tttgtcttcg ctcttttcaa actgggttat tcactgctgc acgtcagtca actaatgctt 60 atcctgacct aagaagctgt gtcaatg 87 <210> 7 <211> 123 <212> Homo DNA sapiens <400> 7 ccattccaga ccagtgtagt cctctgccat gcaatgaaga tggatatatg agctgcaaag 60 atggaaaagc ttcttttaact tgcacttgta aac caggttg gcaaggagaa aagtgtgaat 120 ttg 123 <210> 8 <211> 132 <212> DNA <213> Homo sapiens <400> 8 acataaatga atgcaaagat ccctcaaata taaatggagg ttgcagtcaa atttgtgata 60 atacacctgg aagttaccac tgttcctgta aaaatggttt tgttatgctt tcaaataaga 120 aagattgtaa ag 132 <210> 9 <211 > 126 <212> DNA <213> Homo sapiens <400> 9 atgtggatga atgctctttg aagccaagca tttgtggcac agctgtgtgc aagaacatcc 60 caggagattt tgaatgtgaa tgccccgaag gctacagata taatctcaaa tcaaagtctt 120 gtgaag 126 <210> 10 <211> 122 <212> DNA <213> Homo sapiens <400 > 10 atatagatga atgctctgag aacatgtgtg ctcagctttg tgtcaattac cctggaggtt 60 acacttgcta ttgtgatggg aagaaaggat tcaaacttgc ccaagatcag aagagttgtg 120 ag 122 <210> 11 <211> 116 <212> DNA <213> Homo sapiens <400> 11 gttgtttcag tgtgccttcc cttgaacctt gacacaaagt atgaattact ttacttggcg 60 gagcagtttg caggggttgt tttatattta aaatttcgtt tgccagaaat cagcag 116 <210> 12 <211> 190 <212> DNA <213> Homo sapiens <400> 12 attttcagca gaatttgatt tccggacata tgattcagaa ggcgtgatac tgtacgcaga 60 atctatcgat cactcagcg t ggctcctgat tgcacttcgt ggtggaaaga ttgaagttca 120 gcttaagaat gaacatacat ccaaaatcac aactggaggt gatgttatta ataatggtct 180 atggaatatg 190 <210> 13 <211> 68 <212> DNA <213> Homo sapiens <400> 13 gtgtctgtgg aagaattaga acatagtatt agcattaaaa tagctaaaga agctgtgatg 60 gatataaa 68 <210> 14 < 211> 169 <212> DNA <213> Homo sapiens <400> 14 taaacccctc gtctagatgg atgtatacga agctggaatt tgatgaagca aggagcttct 60 ggaataaagg aaattattca agaaaaacaa aataagcatt gcctggttac tgtggagaag 120 ggctcctact atcctggttc tggaattgct caatttcaca tagattata 169 <210> 15 <211> 152 <212> DNA <213 > Homo sapiens <400> 15 ataatgtatc cagtgctgag ggttggcatg taaatgtgac cttgaatatt cgtccatcca 60 cgggcactgg tgttatgctt gccttggttt ctggtaacaa cacagtgccc tttgctgtgt 120 ccttggtgga ctccacctct gaaaaatcac ag 152 <210> 16 <211> 226 <212> DNA <213> Homo sapiens <400> 16 gatattctgt tatctgttga aaatactgta atatatcgga tacaggccct aagtctatgt 60 tccgatcaac aatctcatct ggaatttaga gtcaacagaa acaatctgga gttgtcgaca 120 ccacttaaaa tagaaaccat ctcccatgaa gaccttcaa a gacaacttgc cgtcttggac 180 aaagcaatga aagcaaaagt ggccacatac ctgggtggcc ttccag 226 <210> 17 <211> 1384 <212> DNA <213> Homo sapiens <400> 17 atgttccatt cagtgccaca ccagtgaatg ccttttataa tggctgcatg gaagtgaata 60 ttaatggtgt acagttggat ctggatgaag ccatttctaa acataatgat attagagctc 120 actcatgtcc atcagtttgg aaaaagacaa agaattctta aggcatcttt tctctgctta 180 taataccttt tccttgtgtg taattatact tatgtttcaa taacagctga agggttttat 240 ttacaatgtg cagtctttga ttattttgtg gtcctttcct gggattttta aaaggtcctt 300 tgtcaaggaa aaaaattctg ttgtgatata aatcacagta aagaaattct tacttctctt 360 gctatctaag aatagtgaaa aataacaatt ttaaatttga atttttttcc tacaaatgac 420 agtttcaatt tttgtttgta aaactaaatt ttaattttat catcatgaac tagtgtctaa 480 atacctatgt ttttttcaga aagcaaggaa gtaaactcaa acaaaagtgc gtgtaattaa 540 atactattaa tcataggcag atactatttt gtttatgttt ttgttttttt cctgatgaag 600 gcagaagaga tggtggtcta ttaaatatga attgaatgga gggtcctaat gccttatttc 660 aaaacaattc ctcaggggga acagctttgg cttcatcttt ctcttgtgtg gcttcacatt 720 taaaccagta tctttattg a attagaaaac aagtgggaca tattttcctg agagcagcac 780 aggaatcttc ttcttggcag ctgcagtctg tcaggatgag atatcagatt aggttggata 840 ggtggggaaa tctgaagtgg gtacattttt taaattttgc tgtgtgggtc acacaaggtc 900 tacattacaa aagacagaat tcagggatgg aaaggagaat gaacaaatgt gggagttcat 960 agttttcctt gaatccaact tttaattacc agagtaagtt gccaaaatgt gattgttgaa 1020 gtacaaaagg aactatgaaa accagaacaa attttaacaa aaggacaacc acagagggat 1080 atagtgaata tcgtatcatt gtaatcaaag aagtaaggag gtaagattgc cacgtgcctg 1140 ctggtactgt gatgcatttc aagtggcagt tttatcacgt ttgaatctac cattcatagc 1200 cagatgtgta tcagatgttt cactgacagt ttttaacaat aaattctttt cactgtattt 1260 tatatcactt ataataaatc ggtgtataat tttaaaatgc atgtgaatat ctttattata 1320 tcaactgttt gaataaaaca aaattacata atagacattt aactcttcaa aaaaaaaaaa 1380 aaaa 1384 <210> 18 <211> 96 <212> DNA <213> Homo sapiens <400> 18 tttgtttata ttttagaaat gattttatat acaaccgtgc atgcatttct gtattggtcg 60 gctttctgg atgcaatttt ttctattcta tatgct 96 <210> 19 <211> 19 <212> RNA <213> Homo sapiens <400> 19 uauuccaga a gcuccuugc 19 <210> 20 <211> 19 <212> RNA <213> Homo sapiens <400> 20 uuugugucaa gguucaagg 19 <210> 21 <211> 19 <212> RNA <213> Homo sapiens <400> 21 auugacacag cuucuuagg 19 <210> 22 <211> 19 <212> RNA <213> Homo sapiens <400> 22 uauaucugua gccuucggg 19 <210> 23 <211> 19 <212> RNA <213> Homo sapiens <400> 23 aaccucacaa cucuucuga 19 < 210> 24 <211> 19 <212> RNA <213> Homo sapiens <400> 24 uccaucacag cuucuuuag 19 <210> 25 <211> 19 <212> RNA <213> Homo sapiens <400> 25 auuugcacga cgcuuccua 19 <210> 26 <211> 19 <212> RNA <213> Homo sapiens <400> 26 uugcacaguu cuucgaugc 19 <210> 27 <211> 19 <212> RNA <213> Homo sapiens <400> 27 uauguggcca cuuuugcuu 19 <210> 28 < 211> 19 <212> RNA <213> Homo sapiens <400> 28 uuuucaaaga ccucccugg 19 <210> 29 <211> 19 <212> RNA <213> Homo sapiens <400> 29 uuccagac accauauuc 19 <210> 30 <211> 19 <212> RNA <213> Homo sapiens <400> 30 auauucacuu ccaugcagc 19 <210> 31 <211> 19 <212> RNA <213> Homo sapiens <400> 31 aagcugagca cacauguuc 19 <210> 32 <211> 19 < 212> R NA <213> Homo sapiens <400> 32 uugacugcaa ccuccauuu 19 <210> 33 <211> 19 <212> RNA <213> Homo sapiens <400> 33 uucugcugaa aaucugcug 19 <210> 34 <211> 19 <212> RNA < 213> Homo sapiens <400> 34 aaucuuucca ccacgaagu 19 <210> 35 <211> 19 <212> RNA <213> Homo sapiens <400> 35 uugguuucca gcaauccau 19 <210> 36 <211> 19 <212> RNA <213> Homo sapiens <400> 36 uagacuuagg gccuguauuc 19 <210> 37 <211> 19 <212> RNA <213> Homo sapiens <400> 37 cuuugcagcu cauauaucc 19 <210> 38 <211> 19 <212> RNA <213> Homo sapiens <400> 38 ugcuuucauu gcuuugucc 19 <210> 39 <211> 19 <212> RNA <213> Homo sapiens <400> 39 ucacuuucca cuuuccgag 19 <210> 40 <211> 19 <212> RNA <213> Homo sapiens <400 > 40 uuugacugca accuccauu 19 <210> 41 <211> 19 <212> RNA <213> Homo sapiens <400> 41 auucucuggg auguucuug 19 <210> 42 <211> 19 <212> RNA <213> Homo sapiens <400> 42 aauagcaagu guaaccucc 19 <210> 43 <211> 19 <212> RNA <213> Homo sapiens <400> 43 uaucacgccu ucugaauca 19 <210> 44 <211> 19 <212> RNA <213> Homo sapiens <400> 44 ugcuucauc a aauuccagc 19 <210> 45 <211> 19 <212> RNA <213> Homo sapiens <400> 45 uuauuccaga agcuccuug 19 <210> 46 <211> 19 <212> RNA <213> Homo sapiens <400> 46 uguucucaga gcauucauc 19 <210> 47 <211> 19 <212> RNA <213> Homo sapiens <400> 47 uaaccuccag gguaauuga 19 <210> 48 <211> 19 <212> RNA <213> Homo sapiens <400> 48 aauccuuucu ucccaucac 19 <210> 49 <211> 19 <212> RNA <213> Homo sapiens <400> 49 aacucuucug aucuugggc 19 <210> 50 <211 > 19 <212> RNA <213> Homo sapiens <400> 50 uucacuuucc acuuuccga 19 <210> 51 <211> 19 <212> RNA <213> Homo sapiens <400> 51 aaaaucuccu gggauguuc 19 <210> 52 <211> 19 <212> RNA <213> Homo sapiens <400> 52 guucucagag cauucaucu 19 <210> 53 <211> 19 <212> RNA <213> Homo sapiens <400> 53 auccuuucuu cccaucaca 19 <210> 54 <211> 19 <212 > RNA <213> Homo sapiens <400> 54 aguaucacgc cuucugaau 19 <210> 55 <211> 19 <212> RNA <213> Homo sapiens <400> 55 acaccauauu ccauagacc 19 <210> 56 <211> 19 <212> RNA <213> Homo sapiens <400> 56 uccacagaca ccauauucc 19 <210> 57 <211> 19 <212> RNA <213> Homo sapiens <400> 57 ucuuccacag acaccauau 19 <210> 58 <211> 19 <212> RNA <213 > Homo sapiens <400> 58 uaauucuucc acagacacc 19 <210> 59 <211> 19 <212> RNA <213> Homo sapiens <400> 59 guucacuuuc cacuuuccg 19 <210> 60 <211> 19 <212> RNA <213> Homo sapiens <4 00> 60 aauuccagaa ccaggauag 19 <210> 61 <211> 19 <212> RNA <213> Homo sapiens <400> 61 aagggcacug uguuguuac 19 <210> 62 <211> 19 <212> RNA <213> Homo sapiens <400> 62 cuggcuucuu cuuuauugc 19 <210> 63 <211> 19 <212> RNA <213> Homo sapiens <400> 63 ucccuggcuu cuucuuuau 19 <210> 64 <211> 19 <212> RNA <213> Homo sapiens <400> 64 uuuccaucuu ugcagcuca 19 <210> 65 <211> 19 <212> RNA <213> Homo sapiens <400> 65 agcuuuucca ucuuugcag 19 <210> 66 <211> 19 <212> RNA <213> Homo sapiens <400> 66 uucugcguac aguaucacg 19 <210> 67 <211> 19 <212> RNA <213> Homo sapiens <400> 67 auaacaucac cuccaguug 19 <210> 68 <211> 19 <212> RNA <213> Homo sapiens <400> 68guauacuuug guuuccagc 19

Claims (11)

An siRNA against protein S for use in a method of treating hemophilia comprising nucleotides of a sequence selected from the group consisting of SEQ ID NO: 028, SEQ ID NO: 029, SEQ ID NO: 030, SEQ ID NO: 049, and SEQ ID NO: 056. The siRNA of claim 1, wherein the siRNA comprises nucleotides of a sequence selected from SEQ ID NO: 028 and SEQ ID NO: 030. delete delete delete delete The siRNA according to claim 1 or 2, wherein the siRNA is directed against an intronic sequence of protein S. The siRNA according to claim 1 or 2 for use in a method of treating hemophilia A. The siRNA according to claim 1 or 2 for use in a method of treating hemophilia B. Administering a molecule comprising the siRNA according to claim 1 or 2 to a non-human subject
Including, a method for treating hemophilia. A pharmaceutical agent for preventing or treating hemophilia, comprising a molecule comprising the siRNA according to claim 1 or 2.

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